Fluorescent dyes, when bonded to target molecules such as proteins, are commonly used to probe the properties of living cells. In particular, such dyes are often used to probe the pH of individual cell compartments. This is commonly achieved by comparing the intensity of fluorescence of a particular dye when it is within a particular cell compartment with a calibration curve of intensity versus pH for that dye.
In some instances, for example when greater accuracy is required, two dyes (with greatly different responses at the measured pH) are used. The ratio of the two observed intensities is compared to a calibration curve, to determine a pH value more reliably.
It has now been realised that even greater accuracy can be achieved by tailoring the most sensitive region of a dye's pH response to the expected pH of the cell or cell compartment. This can be done by using a dye whose pKa is approximately equal to the expected pH of the cell or cell compartment.
At present, long wavelength, fluorescent dyes bonded to suitable target proteins have pKa's of about 6 and above. However, some cell compartments of interest have a pH of less than 6. It would therefore be desirable to provide dyes which have pKa's of less than 6 when bound to protein, thus allowing their pKa to be matched to the approximate pH of their target cell compartment.